A new cell type has been very recently created with clear relevance for therapeutic transplantation into patients with Parkinson's disease (PD). Fibroblasts derived from human skin have been induced to become neurons, and some of these cells can be differentiated into functional dopaminergic neurons, the cell type whose loss leads to most of the motor symptoms of PD. This direct trans-differentiation process does not require the creation of an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC), cell types with a risk of tumor formation (teratoma) after long term transplantation into the brain. The strategy behind this achievement is the identification of transcription factors associated with key developmental events, and the transduction of human fibroblasts with several lentiviral vectors containing the genes for the selected factors. However this strategy also raises potential safety issues which must be resolved prior to clinical trials. These viral vectors integrate into the genome of the hos cells with the potential for tumor induction. The long term effects of sustained and unregulated expression of these multi-functional and potent genes are also unknown. Alternatives to multiple lentiviral vectors for inducing trans-differentiation of somatic cells are under active investigatin. Our proposed project will assess the delivery of transcription factor protein as an alternative to viral vector based transcription factor gene delivery in the induction of dopaminergic differentiation of human fibroblasts. Our group is one of few with experience in the production of neural transcription factor protein modified to include a small peptide (cell penetrating peptide o CPP) to allow the protein to enter target cells, through a process, known as protein transduction. Such a process is particularly suited for this setting where multiple factors need to be delivered in sequence or in combination, with each purified factor added to the culture medium in a manner analogous to current use of natural growth factors. We propose to produce CPP linked forms of the transcription factors Lmx1a and FoxA2, two factors that have been shown to be involved in the dopaminergic differentiation of neurons induced form human fibroblasts. We will determine the extent to which delivery of these modified transcription factors via protein transduction can promote the dopaminergic differentiation of fibroblast derived induced neurons. We will directly compare dopaminergic differentiation driven by CPP linked factors to that seen with lentiviral vector transduction. Cells will be assessed using immuno-histochemical methods to detect and quantitate expression proteins associated with neuronal and dopaminergic differentiation, virtually never seen in normal fibroblasts. The major limitation of CCP based delivery is the very low efficiency of passage of active protein into the free cytosol of the cells where it can then access nuclear gene targets for transcription factors. Most of the presented protein is engulfed and trapped in endocytic vesicles. Our laboratory is one of few actively investigating methods to overcome this limitation. A promising method is photochemical internalization (PCI) where a photosensitizing molecule and appropriate wavelength of light results in release of CPP-linked proteins from membrane enclosed vesicles. We will determine the extent to which the efficiency of protein transduction based delivery of transcription factors and resulting dopaminergic differentiation of fibroblast derived induced neurons can be enhanced through the application of PCI. In this manner we will address two major issues that currently limit the potential clinical translation of this promising new cell type for transplantaton based therapy for Parkinson's disease.